The hepatocyte growth factor receptor (also known as Met) is a tyrosine kinase and is the product of the c-met proto-oncogene. It consists of a 50 kDa α-subunit and of a 145 kDa β-subunit, which are linked by a disulfide bond, the α-subunit being completely extracellular, while the β-subunit includes (from N- to C-terminus) an extracellular region, a transmembrane domain and a cytoplasmic tyrosine kinase domain. The mature α/β hetero-dimeric receptor is generated by proteolytic processing and terminal glycosilation from a 170 kDa single-chain precursor.
HGF, also known as Scatter Factor, is a heparin-binding glycoprotein with a broad spectrum of biological activities including cell proliferation, motility, survival and morphogenesis. It is synthesized and secreted as an inactive single chain precursor (pro-HGF) that is stored into the extracellular matrix due to its high affinity for proteoglycans. Pro-HGF undergoes proteolytic cleavage at residues R494-V495 to give rise to the biologically active form, a disulfide-linked α/β hetero-dimer, where the α-chain consists of an N-terminal domain followed by four kringle domains and the β-chain shares structural homology with the chymotrypsin family of serine proteases. The β-chain, however, lacks proteolytic activity since two of the three critical residues that form the catalytic triad typical of serine proteases are not conserved in HGF. Despite its inability to signal, pro-HGF binds to Met at high affinity and displaces active HGF.
Recently, a number of structure-function studies have shed some light onto the interactions between the extracellular portion of Met and HGF.
The Net extracellular region has a modular structure, which encompasses three functional domains: the Sema domain (present also in Semaphorins and plexins) which spans the first 500 residues at the N-terminus of the protein and has a seven-bladed β-propeller structure, the PSI domain (also found in Plexins, Semaphorins and Integrins) which covers about 50 residues and contains four conserved disulfide bonds, and additional 400 residues which link the PSI domain to the trans-membrane helix and are occupied by four IPT domains (Immunoglobulin-related domains present in Plexins and Transcription factors).
HGF is a bivalent ligand, containing a high affinity binding site for Met in the α-chain and a low affinity binding site in the β-chain. Cooperation between the α- and the β-chain is required for the biological activity of HGF; while the α-chain, and more precisely the N-domain and the first kringle, is sufficient for Met binding, the β-chain is necessary for Met activation.
Resolution of the crystal structure of the SEMA and PSI domains of Met in complex with the β-chain of HGF (see i.a. WO-A-2005/108424) revealed that the low affinity binding site for HGF is located in blades 2-3 of the β-propeller, and that the portion of HGF-β that binds to Met is the same region that serine proteases use to bind to their substrates or inhibitors. Importantly, determination of HGF β-chain crystal structure at 2.53 Å resolution and specific mutagenesis analysis unveiled that the residues involved in Met binding in the activation pocket of HGF β-chain get exposed only following proteolytic conversion of pro-HGF, thus explaining why pro-HGF binds to Met at high affinity without activating it. While the low affinity interaction between the β-chain of HGF and the Sema domain of Met is well characterized both structurally and functionally, at the moment it is not clear what region of Met binds to the α-chain of HGF at high affinity. Thus, the main mechanism by which HGF activates Met still remains poorly understood. This is somehow surprising when considering the great biological and therapeutic importance of this pathway.
HGF-Met signaling is essential during embryogenesis and in tissue regeneration in the adult life. Importantly, deregulated HGF-Met signaling plays a key role in tumorigenesis and metastasis. Inappropriate Met activation by different mechanisms including autocrine HGF stimulation, receptor overexpression, gene amplification and point mutation is described in a wide variety of human malignancies and correlates with poor prognosis. These findings resulted in a growing interest in the HGF-Met pathway as a target for cancer therapy, leading to the development of a variety of Met/HGF inhibitors. These include small molecule compounds targeting Met kinase activity, neutralizing anti-Met or anti-HGF antibodies, decoy receptors and HGF-derived factors. Nevertheless, whether such molecules target the high affinity binding site for HGF and which is the exact molecular mechanism at the basis of HGF binding to Met at high affinity, remains still unknown. This may prevent from isolating more selective therapeutic agents, with increased sensitivity and fewer side effects. The exact knowledge of the Met high affinity binding site for HGF will certainly help to design highly specific antagonists of Met.